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Chromatography Notes
Definition & History
• Chromatography: Technique for separating components of a mixture based on their distribution
between a stationary phase and a mobile phase.
• Makes use of difference in size, binding affinity, charge and other properties to separate material.
• Derived from Greek: "chroma" = color, "graphy" = to measure.
• Michael Tswett (1872–1919):
○ Invented chromatography in 1906.
○ Used calcium carbonate as the stationary phase to separate chlorophyll from other plant
pigments.
Principle of Chromatography
• Based on the partitioning of solutes between two phases.
• Solutes have different solubility or partition coefficients, causing differential movement.
• Adsorption chromatography relies on the interaction of solutes with a solid support (stationary
phase) and their elution in the mobile phase.
• Components with stronger interaction with the stationary phase move slower; weaker interaction
results in faster movement.
• The retention factor (Rf) quantifies the movement of solutes relative to the mobile phase.
Key Contributors
• Richard Kuhn (1900–1967)
○ Studied carotenoids and vitamin B2 (riboflavin).
○ Used chromatography for biochemical separations.
• Archer John Porter Martin (1910–2002)
○ Developed partition chromatography, which led to modern gas chromatography (GC) and
high-performance liquid chromatography (HPLC).
○ Improved gas-liquid chromatography (GLC) for biochemical analysis.
Chromatographic Terminology
• Development: Movement of the mobile phase through the stationary phase.
• Elution: Removal of adsorbed solutes from the stationary phase.
• Eluent: Fluid entering the column.
• Eluate: Fluid exiting the column, containing separated solutes.
• Effluent: Any solvent passing out of the column.
• Retention Volume: Volume of the mobile phase required before solutes begin elution.
• Retention Time (Rt): Time taken for a solute to exit the column.
• Analyte: Mixture being separated and analyzed.
Theoretical Basis of Chromatography
1. Partitioning
○ Components distribute between stationary and mobile phases based on solubility.
○ Mobile Phase: Moves through the stationary phase, dissolving the sample components to
varying degrees.
○ Stationary Phase: Remains fixed and interacts with components based on their chemical
nature.
2. Adsorption
○ Some components adhere to the stationary phase more strongly than others.
○ Higher affinity → slower movement; lower affinity → faster movement.
3. Partition Coefficient (K)
○ Describes the relative affinity of a solute for the stationary vs. mobile phase.
Higher K → stronger interac on with sta onary phase → slower movement.
unit 2 Page 1
, ○ Higher K → stronger interac on with sta onary phase → slower movement.
○ Lower K → stronger interac on with mobile phase → faster movement.
4. Retention Factor (Rf)
○ Rf = Distance moved by compound / Distance moved by mobile phase.
○ Helps identify compounds based on their movement in thin-layer chromatography (TLC).
Chromatographic Process
1. Column packed with silica gel (stationary phase).
2. Analyte loaded onto the column.
3. Mobile phase (solvent) added; components start separating.
4. Components move based on polarity:
○ Nonpolar mobile phase (hexane) → polar compounds adhere to silica.
○ Polar mobile phase (acetonitrile) → polar compounds dissolve and elute.
Types of Chromatography
1. Planar Chromatography
○ Paper Chromatography: Separation based on partitioning between paper fibers and solvent.
○ Thin Layer Chromatography (TLC): Uses a thin layer of adsorbent (silica/alumina) for
separation.
2. Column Chromatography (gas column packed with silica in an organic solvent, reaction mixture
loaded on the silica bed with the help of glass pipette)
○ Adsorption Chromatography: Components separate based on adhesion to solid phase.
○ Partition Chromatography: Separation based on partitioning between liquid stationary and
mobile phases.
○ Ion Exchange Chromatography: Uses charged stationary phase to separate ions.
○ Affinity Chromatography: Separation based on specific binding interactions.
○ Gel Chromatography: Size-exclusion technique for separating biomolecules.
○ Gas-Liquid Chromatography (GLC): Uses a gas as the mobile phase.
○ High-Performance Liquid Chromatography (HPLC): High-pressure liquid phase for efficient
separation.
unit 2 Page 2
Chromatography Notes
Definition & History
• Chromatography: Technique for separating components of a mixture based on their distribution
between a stationary phase and a mobile phase.
• Makes use of difference in size, binding affinity, charge and other properties to separate material.
• Derived from Greek: "chroma" = color, "graphy" = to measure.
• Michael Tswett (1872–1919):
○ Invented chromatography in 1906.
○ Used calcium carbonate as the stationary phase to separate chlorophyll from other plant
pigments.
Principle of Chromatography
• Based on the partitioning of solutes between two phases.
• Solutes have different solubility or partition coefficients, causing differential movement.
• Adsorption chromatography relies on the interaction of solutes with a solid support (stationary
phase) and their elution in the mobile phase.
• Components with stronger interaction with the stationary phase move slower; weaker interaction
results in faster movement.
• The retention factor (Rf) quantifies the movement of solutes relative to the mobile phase.
Key Contributors
• Richard Kuhn (1900–1967)
○ Studied carotenoids and vitamin B2 (riboflavin).
○ Used chromatography for biochemical separations.
• Archer John Porter Martin (1910–2002)
○ Developed partition chromatography, which led to modern gas chromatography (GC) and
high-performance liquid chromatography (HPLC).
○ Improved gas-liquid chromatography (GLC) for biochemical analysis.
Chromatographic Terminology
• Development: Movement of the mobile phase through the stationary phase.
• Elution: Removal of adsorbed solutes from the stationary phase.
• Eluent: Fluid entering the column.
• Eluate: Fluid exiting the column, containing separated solutes.
• Effluent: Any solvent passing out of the column.
• Retention Volume: Volume of the mobile phase required before solutes begin elution.
• Retention Time (Rt): Time taken for a solute to exit the column.
• Analyte: Mixture being separated and analyzed.
Theoretical Basis of Chromatography
1. Partitioning
○ Components distribute between stationary and mobile phases based on solubility.
○ Mobile Phase: Moves through the stationary phase, dissolving the sample components to
varying degrees.
○ Stationary Phase: Remains fixed and interacts with components based on their chemical
nature.
2. Adsorption
○ Some components adhere to the stationary phase more strongly than others.
○ Higher affinity → slower movement; lower affinity → faster movement.
3. Partition Coefficient (K)
○ Describes the relative affinity of a solute for the stationary vs. mobile phase.
Higher K → stronger interac on with sta onary phase → slower movement.
unit 2 Page 1
, ○ Higher K → stronger interac on with sta onary phase → slower movement.
○ Lower K → stronger interac on with mobile phase → faster movement.
4. Retention Factor (Rf)
○ Rf = Distance moved by compound / Distance moved by mobile phase.
○ Helps identify compounds based on their movement in thin-layer chromatography (TLC).
Chromatographic Process
1. Column packed with silica gel (stationary phase).
2. Analyte loaded onto the column.
3. Mobile phase (solvent) added; components start separating.
4. Components move based on polarity:
○ Nonpolar mobile phase (hexane) → polar compounds adhere to silica.
○ Polar mobile phase (acetonitrile) → polar compounds dissolve and elute.
Types of Chromatography
1. Planar Chromatography
○ Paper Chromatography: Separation based on partitioning between paper fibers and solvent.
○ Thin Layer Chromatography (TLC): Uses a thin layer of adsorbent (silica/alumina) for
separation.
2. Column Chromatography (gas column packed with silica in an organic solvent, reaction mixture
loaded on the silica bed with the help of glass pipette)
○ Adsorption Chromatography: Components separate based on adhesion to solid phase.
○ Partition Chromatography: Separation based on partitioning between liquid stationary and
mobile phases.
○ Ion Exchange Chromatography: Uses charged stationary phase to separate ions.
○ Affinity Chromatography: Separation based on specific binding interactions.
○ Gel Chromatography: Size-exclusion technique for separating biomolecules.
○ Gas-Liquid Chromatography (GLC): Uses a gas as the mobile phase.
○ High-Performance Liquid Chromatography (HPLC): High-pressure liquid phase for efficient
separation.
unit 2 Page 2
